Electrochemiluminescence is well-described in U.S. Pat. Nos. 5,061,445; 5,068,088; 5,093,268; 5,147,806; 5,221,605; 5,238,808; 5,247,243; 5,296,191; and 5,310,687. Each of these patents is incorporated herein by reference. Nevertheless, the assay formats themselves (, e.g., for endotoxins) require novel and unobvious approaches to problems in the newly emerging field of electrochemiluminescence.
The only known assay for endotoxins is an FDA-approved assay on the market known as the LAL (Limulus Amebocyte Lysate) Test. This test takes 2 days and the reproducibility of the assay is not very good to the average practitioner. It is desirable to improve the assay so that it is less labor intensive and more reproducible. The present invention overcomes the first problem and has a great potential to overcome the second problem with reproducibility.
Endotoxins or lipopolysaccharides (LPS) are complex, amphiphatic macromolecules that comprise three genetically, biochemically, and antigenically distinct domains referred to as the O-side chain, core oligosaccharide, and lipid A. The LPS core structure, consisting of the core oligosaccharide and lipid A, is relatively conserved among different species. This observation, in addition to the knowledge that many biological activities of LPS reside in the lipid A moiety, is important when considering any modification and derivatization of the LPS molecule.
The present invention protects the lipid A as well as the core oligosaccharide structure, during labeling with the ruthenium (II) tris-bipyridine NHS ester, referred to as the TAG-label hereafter. LPS structures with ethanolamine groups are selected for labeling since they contain amino groups to which the TAG-label reacts. Ethanolamine-containing lipopolysacchrides also have these groups in the biologically important lipid A portion and in the core structure.
The TAG-labeled material is then used to develop a sensitive, reproducible, and fast detection method for endotoxins in aqueous solutions using the ORIGEN.TM. Analyzer to detect the electrochemiluminescence originating from the TAG-label in contact with an electrode in the aforementioned instrument.